Istvan Szabo

Influence of a Probiotic Strain of Enterococcus faecium on Salmonella enterica Serovar Typhimurium DT104 Infection in a Porcine Animal Infection Model

ABSTRACT The beneficial effects of probiotic Enterococcus spp. in different hosts, such as mice and humans, have previously been reported in several studies. However, studies of large domestic animals, as well as challenge studies with pathogenic microorganisms, are very rare. Here, we investigated the influence of oral treatment of pigs with the probiotic bacterium Enterococcus faecium NCIMB 10415 on Salmonella enterica serovar Typhimurium DT104 infections in weaning piglets. Clinical symptoms, fecal excretion, the organ distribution of Salmonella, and the humoral immune response (immunoglobulin G [IgG], IgM, and IgA levels) in serum were examined. A pool of 89 piglets was randomly divided into probiotic and control groups. The probiotic group received a feed supplement containing E. faecium starting on day 14 postpartum prior to challenge with Salmonella serovar Typhimurium DT104 at 28 days postpartum. After challenge with Salmonella serovar Typhimurium DT104, piglets in both groups showed no severe clinical signs of salmonellosis. However, fecal excretion and colonization of Salmonella in organs were significantly greater in piglets fed E. faecium. Likewise, the humoral immune response against Salmonella (serum IgM and IgA levels) was significantly greater in the probiotic group animals than in control animals. The results of this study suggest that E. faecium NCIMB 10415 treatment enhanced the course of infection in weaning piglets challenged with Salmonella serovar Typhimurium DT104. However, the probiotic treatment also appeared to result in greater production of specific antibodies against Salmonella serovar Typhimurium DT104.

Identification of a novel transposon-associated phosphoethanolamine transferase gene, mcr-5, conferring colistin resistance in d-tartrate fermenting Salmonella enterica subsp. enterica serovar Paratyphi B

Objectives Plasmid-mediated mobilized colistin resistance is currently known to be caused by phosphoethanolamine transferases termed MCR-1, MCR-2, MCR-3 and MCR-4. However, this study focuses on the dissection of a novel resistance mechanism in mcr-1-, mcr-2- and mcr-3-negative d-tartrate fermenting Salmonella enterica subsp. enterica serovar Paratyphi B (Salmonella Paratyphi B dTa+) isolates with colistin MIC values >2 mg/L. Methods A selected isolate from the strain collection of the German National Reference Laboratory for Salmonella was investigated by WGS and bioinformatical analysis to identify novel phosphoethanolamine transferase genes involved in colistin resistance. Subsequently PCR screening, S1-PFGE and DNA-DNA hybridization were performed to analyse the prevalence and location of the identified mcr-5 gene. Cloning and transformation experiments in Escherichia coli DH5α and Salmonella Paratyphi B dTa+ control strains were carried out and the activity of MCR-5 was determined in vitro by MIC testing. Results In this study, we identified a novel phosphoethanolamine transferase in 14 mcr-1-, mcr-2- and mcr-3-negative Salmonella Paratyphi B dTa+ isolates with colistin MIC values >2 mg/L that were received during 2011-13. The respective gene, further termed as mcr-5 (1644 bp), is part of a 7337 bp transposon of the Tn3 family and usually located on related multi-copy ColE-type plasmids. Interestingly, in one isolate an additional subclone with a chromosomal location of the mcr-5 transposon was observed. Conclusions Our findings suggest that the transfer of colistin-resistance-mediating phosphoethanolamine transferase genes from bacterial chromosomes to mobile genetic elements has occurred in multiple independent events raising concern regarding their variety, prevalence and impact on public health.

Time course of infection with Salmonella typhimurium and its influence on fecal shedding, distribution in inner organs, and antibody response in fattening pigs.

This is the first longitudinal study conducted over the entire 5-month fattening period in pigs to investigate the infection dynamics of Salmonella Typhimurium and the association between antibody response and the prevalence of these bacteria in feces. A total of 16 weaning pigs were infected with Salmonella Typhimurium DT104 followed by clinical examination and blood and fecal sampling until slaughter 138 days postinoculation. To investigate fecal shedding rates and distribution patterns of Salmonella in internal organs regarding premortem stress, one group of swine was transported before slaughter; the other group was slaughtered without being transported. A positive correlation between bacteremia-associated fever and fecal shedding rate was observed, although 69% (11 of 16) of infected pigs had no diarrhea. All animals excreted Salmonella Typhimurium at high levels within 2 weeks postinoculation; thereafter, the number of positive pigs declined and Salmonella shedding became intermittent. In contrast, the proportion of pigs that tested seropositive was higher over the entire fattening period (except during the first 3 weeks postinoculation), revealing the advantage of enzyme-linked immunosorbent assay for Salmonella screening on herd level. Concerning the distribution in internal organs and cross-contamination during slaughter, the highest level of Salmonella was detected in tonsils and jejunal and ileocecal lymph nodes, whereas salmonellae could not be detected in muscle, spleen, and liver. No specific influence of transport-induced stress on Salmonella shedding rates in feces and distribution patterns in organs was observed.

Comparative examination and validation of ELISA test systems for Salmonella typhimurium diagnosis of slaughtering pigs

The most frequently isolated Salmonella serotype from pork in Germany is S. typhimurium, especially phagetype DT 104. The monitoring programs on Salmonella in swine are based on enzyme-linked immunoadsorbent assay (ELISA) detecting antibodies in serum or meat juice. These serological results are used to classify swine herds in three categories to assess the hygienic status of farm regarding Salmonella infection in pigs. The object of this study was the comparative evaluation of four indirect Salmonella ELISA tests approved in Germany to detect Salmonella typhimurium infection of swine. Three tests (A-C) are based on LPS-antigen and directed against specific IgG-antibodies. The fourth test (D) bases on a whole-cell-lysate antigen and discriminates between Salmonella specific IgA-, IgM- and IgG-antibodies. In a longitudinal study sixteen 6 weeks old weaning pigs were orally infected with S. typhimurium DT 104. During an observation period of 138d clinical and bacteriological parameters were monitored and serum samples obtained at regular intervals as well as meat juice samples taken at slaughter were examined by the respective ELISA systems. Study results reveal that all tested ELISA systems are able to detect S. typhimurium infection in pigs in both sample matrices, blood serum and meat juice whereas test D showed the highest sensitivity to detect Salmonella antibodies in pigs. The sensitivity to detect Salmonella antibodies varied between tests A and C according to the used cut-off (test specific cut-off vs. recommended surveillance cut-off) resulting in a change of seroprevalence and hence may influence the Salmonella status of the farm.

Studies on the effect of an Enterococcus faecium probiotic on T cell populations in peripheral blood and intestinal epithelium and on the susceptibility to Salmonella during a challenge infection with Salmonella Typhimurium in piglets

Although Enterococcus faecium is used as a probiotic feed supplement in animal production, feeding of the bacterium to piglets resulted in a more severe infection with Salmonella Typhimurium DT104 during a challenge experiment. To enlighten the mode of action by which E. faecium affected the piglets’ health, we investigated the influence of the probiotic bacterium on the development of intestinal and circulating immune cells during a challenge experiment with S.Typhimurium DT104. To minimise varying impacts of the maternal immunity on the course of infection, only piglets were implemented that descended from Salmonella-free sows. In addition, the potency of purified blood and intraepithelial immune cells to control the growth of Salmonella was tested in vitro. In animals treated with E. faecium, a reduction of intraepithelial CD8αβ T cells, reduced circulating CD8αβ T cells and a less efficient control of intracellular Salmonella growth, mediated by peripheral blood mononuclear cells, were observed.

VIM-1-producing Salmonella Infantis isolated from swine and minced pork meat in Germany

1 Rasmussen BA, Bush K, Keeney D et al. Characterization of IMI-1 b-lactamase, a class A carbapenem-hydrolyzing enzyme from Enterobacter cloacae. Antimicrob Agents Chemother 1996; 40: 2080–6. 2 Naas T, Dortet L, Iorga BI. Structural and functional aspects of class A carbapenemases. Curr Drug Targets 2016; 17: 1006–28. 3 Di Luca MC, Skaare D, Aasnaes B et al. Identification of a novel IMI carbapenemase variant (IMI-9) in Enterobacter cloacae complex. Int J Antimicrob Agents 2016; 48: 764–5. 4 Aubron C, Poirel L, Ash RJ et al. Carbapenemase-producing Enterobacteriaceae, U.S. rivers. Emerg Infect Dis 2005; 11: 260–4. 5 Dang B, Mao D, Luo Y. Complete nucleotide sequence of pGA45, a 140,698-bp IncFIIY plasmid encoding blaIMI-3-mediated carbapenem resistance, from river sediment. Front Microbiol 2016; 7: 188. 6 Rojo-Bezares B, Martin C, L opez M et al. First detection of blaIMI-2 gene in a clinical Escherichia coli strain. Antimicrob Agents Chemother 2012; 56: 1146–7. 7 Doumith M, Day M, Ciesielczuk H et al. Rapid identification of major Escherichia coli sequence types causing urinary tract and bloodstream infections. J Clin Microbiol 2015; 53: 160–6. 8 Rosenblueth M, Martinez L, Silva J et al. Klebsiella variicola, a novel species with clinical and plant-associated isolates. Syst Appl Microbiol 2004; 27: 27–35. 9 van Veen SQ, Claas EC, Kuijper EJ. High-throughput identification of bacteria and yeast by matrix-assisted laser desorption ionization-time of flight mass spectrometry in conventional medical microbiology laboratories. J Clin Microbiol 2010; 48: 900–7. 10 Maatallah M, Vading M, Kabir MH et al. Klebsiella variicola is a frequent cause of bloodstream infection in the Stockholm area, and associated with higher mortality compared to K. pneumoniae. PLoS One 2014; 9: e113539.

Rapid real-time PCR methods to distinguish Salmonella Enteritidis wildtype field isolates from vaccine strains Salmovac SE/Gallivac SE and AviPro SALMONELLA VAC E

Salmonella enterica serovar Enteritidis is a major non-typhoid Salmonella serovar causing human salmonellosis mainly associated with the consumption of poultry and products thereof. To reduce infections in poultry, S. Enteritidis live vaccine strains AviPro SALMONELLA VAC E and Salmovac SE/Gallivac SE have been licensed and used in several countries worldwide. To definitively diagnose a S. Enteritidis contamination in vaccinated herds a reliable and fast method for the differentiation between vaccine and wildtype field isolates is required. In this study, we developed and validated real-time PCR (qPCR) assays to distinguish those variants genetically. Suitable target sequences were identified by whole genome sequencing (WGS) using the Illumina MiSeq system. SNP regions in kdpA and nhaA proved to be most useful for differentiation of AviPro SALMONELLA VAC E and Salmovac SE/Gallivac SE, respectively, from wildtype strains. For each vaccine strain one TaqMan-qPCR assay and one alternative approach using High Resolution Melting (HRM) analysis was designed. All 30 Salmovac SE and 7 AviPro SALMONELLA VAC E vaccine strain reisolates tested were correctly identified by both approaches (100% inclusivity). Furthermore, all 137 (TaqMan) and 97 (HRM) Salmonella non-vaccine and related Enterobacteriaceae strains tested were excluded (100% exclusivity). The analytical detection limits were determined to be approx. 10(2) genome copies/reaction for the TaqMan and 10(4) genome copies/reaction for the HRM approach. The real-time PCR assays proved to be a reliable and fast alternative to the cultural vaccine strain identification tests helping decision makers in control measurements to take action within a shorter period of time.

In Vivo Transfer and Microevolution of Avian Native IncA/C2 blaNDM-1-Carrying Plasmid pRH-1238 during a Broiler Chicken Infection Study

ABSTRACT The emergence and spread of carbapenemase-producing Enterobacteriaceae (CPE) in wildlife and livestock animals pose an important safety concern for public health. With our in vivo broiler chicken infection study, we investigated the transfer and experimental microevolution of the blaNDM-1-carrying IncA/C2 plasmid (pRH-1238) introduced by avian native Salmonella enterica subsp. enterica serovar Corvallis without inducing antibiotic selection pressure. We evaluated the dependency of the time point of inoculation on donor (S. Corvallis [12-SA01738]) and plasmid-free Salmonella recipient [d-tartrate-fermenting (d-Ta+) S. Paratyphi B (13-SA01617), referred to here as S. Paratyphi B (d-Ta+)] excretion by quantifying their excretion dynamics. Using plasmid profiling by S1 nuclease-restricted pulsed-field gel electrophoresis, we gained insight into the variability of the native plasmid content among S. Corvallis reisolates as well as plasmid acquisition in S. Paratyphi B (d-Ta+) and the enterobacterial gut microflora. Whole-genome sequencing enabled us to gain an in-depth insight into the microevolution of plasmid pRH-1238 in S. Corvallis and enterobacterial recipient isolates. Our study revealed that the fecal excretion of avian native carbapenemase-producing S. Corvallis is significantly higher than that of S. Paratyphi (d-Ta+) and is not hampered by S. Paratyphi (d-Ta+). Acquisition of pRH-1238 in other Enterobacteriaceae and several events of plasmid pRH-1238 transfer to different Escherichia coli sequence types and Klebsiella pneumoniae demonstrated an interspecies broad host range. Regardless of the microevolutionary structural deletions in pRH-1238, the single carbapenem resistance marker blaNDM-1 was maintained on pRH-1238 throughout the trial. Furthermore, we showed the importance of the gut E. coli population as a vector of pRH-1238. In a potential scenario of the introduction of NDM-1-producing S. Corvallis into a broiler flock, the pRH-1238 plasmid could persist and spread to a broad host range even in the absence of antibiotic pressure.

The zoonotic agent Salmonella

Salmonella (S.) Enterica serovars form a group of pathogens that differ widely in their host range within mammals, birds and reptiles. They can differ substantially in clinical manifestations, ranging from an asymptomatic state to severe illness . Serovars can be host-restricted (e.g S. typhi in humans), host-adapted (e.g. S. choleraesuis in pigs and infrequently in humans) and broad range infecting diverse avian and mammalian hosts with a wide range of diseases. Currently, the traditional Salmonella serotyping scheme according to White-Kauffmann-Le Minor is accepted worldwide as the “gold standard” for the classification of Salmonellae below the subspecies level and is widely used in surveillance of the pathogen . The use of serotyping within Salmonella as a typing method is so widely accepted that governmental agencies have formulated guidelines intended to reduce human salmonellosis by identifying the common serovars typhimurium and Enteritidis . The most common vehicles of transmission are meat, meat products, eggs and egg products that contain Salmonella serovars because animals are infected or because fecal contamination occurs during processing . The majority of human cases are caused by only a few non-typhoidal serovars. In 2012, approximately 20,000 cases of non-typhoidal salmonellosis are reported in Germany (http://www3.rki.de//survstat). In 1995 the dominance of only a few serovars is even more pronounced in Germany, where S. enteritidis (61 %) and S. typhimurium (23 %) accounted for more than 80 % of human isolates reported to the NRC at the Robert Koch Institute . In 2012 this percentage for both serovars were reduced to 62 %, each approx. 31 %. Other serovars of different vehicles were found in outbreaks and also serovar analysis showed that the spectrum of single cases in children changed. In this article we focus on the prevalence of Salmonella enterica in animal food and humans and its change of serotypes and subtypes over up to two decades.

Adhesion of Salmonella to Pancreatic Secretory Granule Membrane Major Glycoprotein GP2 of Human and Porcine Origin Depends on FimH Sequence Variation

Bacterial host tropism is a primary determinant of the range of host organisms they can infect. Salmonella serotypes are differentiated into host-restricted and host-adapted specialists, and host-unrestricted generalists. In order to elucidate the underlying molecular mechanisms of host specificity in Salmonella infection, we investigated the role of the intestinal host cell receptor zymogen granule membrane glycoprotein 2 (GP2), which is recognized by FimH adhesin of type 1 fimbriae found in Enterobacteriaceae. We compared four human and two porcine GP2 isoforms. Isoforms were expressed in Sf9 cells as well as in one human (HEp-2) and one porcine (IPEC-J2) cell line. FimH genes of 128 Salmonella isolates were sequenced and the 10 identified FimH variants were compared regarding adhesion (static adhesion assay) and infection (cell line assay) using an isogenic model. We expressed and characterized two functional porcine GP2 isoforms differing in their amino acid sequence to human isoforms by approximately 25%. By comparing all isoforms in the static adhesion assay, FimH variants were assigned to high, low or no-binding phenotypes. This FimH variant-dependent binding was neither specific for one GP2 isoform nor for GP2 in general. However, cell line infection assays revealed fundamental differences: using HEp-2 cells, infection was also FimH variant-specific but mainly independent of human GP2. In contrast, this FimH variant dependency was not obvious using IPEC-J2 cells. Here, we propose an alternative GP2 adhesion/infection mechanism whereby porcine GP2 is not a receptor that determined host-specificity of Salmonella. Salmonella specialists as well as generalists demonstrated similar binding to GP2. Future studies should focus on spatial distribution of GP2 isoforms in the human and porcine intestine, especially comparing health and disease.